Dietary neutral lipid level and source affect food intake, nutrient absorption, gut structure, enzymatic activity and growth in marine fish larvae

Transcription

1 Dietary neutral lipid level and source affect food intake, nutrient absorption, gut structure, enzymatic activity and growth in marine fish larvae S. Morais 1, L.E.C. Conceição 1, W. Koven 2, I. Rønnestad 3, C. Cahu 4, J.L. Zambonino Infante 4, and M.T. Dinis 1 1 CCMAR, Universidade do Algarve, Portugal 2 IOLR, The National Centre for Mariculture, Eilat, Israel 3 Department of Biology, University of Bergen, Norway 4 IFREMER, Station de Brest, France

2 Background Fish larvae Extremely high growth rates high demands for energy and structural components Lipids Important source of metabolic energy Components of biological membranes Precursors of essential metabolites The requirements for essential fatty acids (EFA) have been extensively studied in marine fish larvae Few studies have examined the effect of total lipid levels in larval diets

3 Background Meet larval requirements using poor sources of EFA (predominantly neutral lipids): excessive lipid content imbalanced lipid class composition poor larval growth and performance? High dietary neutral lipid levels several effects can be speculated: Digestion (decrease in efficiency or activity of digestive enzymes) Absorption (reduction in absorption efficiency) Ingestion (regulation of food intake according to dietary energy?)

4 Dietary Triacylglycerol Source and Level Affects Performance and Lipase Expression in Larval Seabass (Dicentrarchus labrax) Morais et al. (24) Lipids 39,

5 Experimental Diet Formulation and Proximate Composition F7.5 T7.5 C7.5 F15 T15 C15 Diet Ingredients (g/kg dry diet) Constant ingredients Fish meal Hydrolyzed fish meal Soybean lecithin Vitamin mixture Mineral mixture Betaine Variable ingredients Fish oil (cod liver oil) Triolein (purified) Coconut oil Starch Proximate Composition Dry matter (DM, %) 97.8±. 97.6±. 97.6± ±. 97.4± ±. Protein (N x 6.25) (% DM) 58.3± ± ± ±. 57.7±. 58.4±.4 Lipid (% DM) 17.± ±.7 16.± ± ± ±.7 Ash (% DM) 12.±.1 12.±. 12.2±.1 12.±. 13.5±.1 12.±. Protein energy / Lipid energy

6 Lipase specific activity (U/mg protein DAH a ab a ab b a Lipase specific activity Source of dietary lipid Level of dietary lipid. F7.5 T7.5 C7.5 F15 T15 C15 Fish oil: age-dependent effect 6 52 DAH Lipase activity (U/mg protein) a b c a b bc 1 F7.5 T7.5 C7.5 F15 T15 C15

7 Wet weight (mg Growth, 52 DAH ac a ac bc c a Growth Not related to lipase specific activity Lipase activity not limiting for growth? F7.5 T7.5 C7.5 F15 T15 C15 6 Lipase activity, 52 DAH Lipase activity (U/mg protein) a b c a b bc 1 F7.5 T7.5 C7.5 F15 T15 C15

8 Conclusions The results suggest the existence of a regulatory mechanism of neutral lipolytic activity and secretion according to the FA composition of the diet The understanding of the underlying mechanisms controlling such and adaptation is still scarce several hypothesis: Specificity of lipase towards FA differing in chain length and degree of saturation; Endocrine factors; chemical nature of FA (particularly chain length) affects CCK secretion in humans and other mammals;

9 Conclusions However growth was not related to lipase activity the two factors might be independent (possibly a secretion in excess to dietary needs) Physiological consequence of such a regulation? For fish oil, the effect was age-dependent not only nutritional requirements but also digestive function may show important changes between early larvae and later larvae/juvenile stages

10 Growth, gut histology and absorption of fatty acids and amino acids in response to dietary neutral lipid level in Senegalese sole (Solea senegalensis Kaup 1858) larvae Morais et al. (25) British Journal of Nutrition 93, Morais et al. (25) Aquaculture 246,

11 Diets: - Non-enriched Artemia (NEA); - Artemia enriched on a soybean oil emulsion (EA). 4 4 NEA % Dry Weight % Dry Weight EA Total Protein Total Lipid Total Protein Total Lipid Experiment I (FA absorp.) Experiment II (AA absorp.)

12 Growth 8 7 * NEA EA Dry weigth (mg) NS Experiment I (FA absorp.) 34 DAH Experiment II (AA absorp.) 36 DAH Non-enriched Artemia (NEA): better larval growth, compared to Artemia enriched on a soybean oil emulsion (EA), a diet containing a higher neutral lipid level

13 Histology of the gut epithelium (34 DAH) NEA EA Artemia enriched on soybean oil (EA) induced a much higher accumulation of lipid droplets within the gut enterocytes Can this accumulation function as a physical barrier to efficient lipid absorption?

14 Lipid absorption Tube feeding (32 DAH): 14 C-Triolein (TRI), a triacylglycerol 14 C- Phosphatidylcholine 1,2 di-oleoyl (PC), a phospholipid 14 C- Oleic acid (OA), a free fatty acid Total absorbed label (% total fed) Absorption NEA EA TRI PC OA Significantly lower label absorption in larvae fed EA Lipid accumulation barrier for FA absorption?

15 Lipid absorption Tube feeding: Free FA (32 DAH) 14 C- Oleic acid (OA), 18:1(n-9) 14 C- Stearic acid (SA), 18: 14 C- Docosahexaenoic acid (DHA), 22:6(n-3) Absorption NEA Total absorbed label (% total fed) * EA OA SA DHA Absorption of lipids differing in FA composition may not be equally affected by lipid droplet accumulation in the enterocytes

16 Protein and amino acid absorption Larvae fed on Artemia radiolabelled with 14 C-AA [U- 14 C] Protein hydrolisate Label in water (% total label fed) Evacuation into water NEA EA * 1 h 3 h 24 h Lipid inclusions within the enterocytes do not appear to affect total AA absorption efficiency

17 Protein and amino acid absorption Larvae fed on Artemia radiolabelled with 14 C-AA [U- 14 C] Protein hydrolisate Label in water (% total label fed) Evacuation into water NEA EA * 1 h 3 h 24 h Faster AA absorption in larvae fed NEA higher food intake of NEA (related to gut transit time)

18 Conclusions The long term feeding of a diet higher in neutral lipid may affect the capacity of larvae to efficiently absorb dietary FA lipid droplet accumulation in the enterocytes may function as a barrier for FA but not for AA absorption Lipid level in the diet may affect the rate of AA absorption and, consequently, evacuation rates (effects on food intake??) Accumulation of lipid droplets in the enterocytes may not be an obstacle for the efficient utilization of EFA (long chain PUFA - higher specificity of lipases, affinity of FABP and rate of esterification)

19 Food intake and absorption are affected by dietary lipid level and lipid source in seabream (Sparus aurata L.) larvae Morais et al. (25), submitted

20 1 st Experiment: Artemia enriched in high and low doses of fish oil emulsion Seabream, 34 DAH 2. NS Larval DW (mg) HF LF LF diet met minimum EFA requirements for growth

21 Cold chase trial, at 33 DAH, with 14 C-Artemia ( 14 C-OA liposomes) Gut Body 6 LF 16 LF No. Artemia/larva HF No. Artemia/larva HF 2: 3: 3:3 4: 5: 2: 3: 3:3 4: 5: Time after feeding (hours) Time after feeding (hours) HF significantly higher ingestion but lower nutrient absorption Inverse relationship between food intake and absorption efficiency (more rapid passage through the gut) A higher neutral lipid level did not decrease larval food intake, as observed in older fish

22 2 nd Experiment: Co-feeding Artemia enriched in high and low doses of soybean oil emulsion + high or low lipid microdiet (MD) Seabream, 34 DAH Larval DW (mg) *. HS LS Higher lipid diet (HS) significantly lower growth

23 Cold chase trial, at 32 DAH, with MD containing 14 C-OA Gut Body 18 LS 7 LS ug microdiet/larva HS ug microdiet/larva HS 2: 3: 3:3 4: 5: 2: 3: 3:3 4: 5: Time after feeding (hours) Time after feeding (hours) HS significantly lower ingestion and lower FA absorption The total neutral lipid level of the diet was inversely correlated with food intake

24 Conclusions In fish larvae food intake may not be regulated by dietary total lipid level and FA composition possibly has a more important role in controlling ingestion Possible explanations might be: FA digestibility, by influencing digestive and absorption efficiency, may affect the rate of gut clearance and subsequent refeeding; Palatability (mostly with microdiets); FA stimulation of release of gastrointestinal hormones (e.g. CCK);

25 General conclusions

26 General conclusions Existing data seems to collectively indicate that enzymatic capacity is not a limiting factor for fish larvae to grow well on high lipid diets The main obstacle to high neutral lipid inclusion levels in diets for marine fish larvae may be at the absorption level Seabream larvae do not simply regulate their food intake according to total lipid level (i.e., energy content) and other factors must be involved

27 Take Home Message Lipid level in diets for marine fish larvae may have an important impact in several factors influencing growth and development but it clearly cannot be dissociated of its FA composition, which appears to play a central role on the physiological impacts of dietary lipid, at the ingestion, digestion and absorption levels

28 Acknowledgements Fundação para a Ciência e a Tecnologia, Portugal Associação Viver a Ciência Citomed